Immobilized carbohydrate biosensor

ABSTRACT

A biosensor in which a carbohydrate or a derivative of a carbohydrate is used to generate a detectable signal by way of the specific binding to a protein, a virus or a cell.

REFERENCE TO A RELATED APPLICATION

This is a continuation application of U.S. patent application Ser. NO.09/766,659 filed Jan. 23, 2001 which in turn is a continuation of U.S.patent application Ser. No. 08/356,229 filed Dec. 19, 1994, now U.S.Pat. No. 6,231,733 which in turn is a continuation of PCT/SE94/00343filed Apr. 18, 1994 which claims the benefit of Swedish prioritydocument 9301270-6 filed Apr. 19, 1993, all of which are relied on andincorporated herein by reference.

The present invention relates to a biosensor in which a carbohydrate ora derivative thereof is used to generate a detectable signal via thespecific binding of a protein, a virus or a cell.

BACKGROUND

Biosensors are characterised by a physical or chemical signaltransducer, which response is activated by a specific interactionbetween a biochemical structure (which directly or indirectly has beenbound to the transducer) and one or several analytes.

Biosensors are used to detect the analyte/analytes and in certain casesalso for quantification of the analyte/analytes.

The advantages of the biosensor are that a physical or chemicaltransducer has been made specific so that a general physical or chemicalparameter (e.g. temperature, pH, optical density) can be used for thedetection of one specific substance in a complex mixture of non-specificsubstances.

The limitations of the biosensor are the specificity of the biochemicalstructure bound to the transducer, the range of specificity andstability, and, that the transducer signal has to be made independent ofthe background changes in the parameter that the transducer ismeasuring. In Methods of Enzymology, volume 137, several articles aredescribing different aspects of biosensors.

DEFINITIONS

Biosensor-physical or chemical signal transducer, e.g. photometer,chemical electrode, temperature or pressure signal transducer, whichdirectly or indirectly has been connected with a biochemical structure.In previous biosensors one has preferentially used an enzyme, a specificprotein or antibody as the biochemical structure and in this way thebiosensors have been given the property of being able to detectsubstances which specifically bind to the biochemical structure in aqualitative or quantitative way.

Reflection measurement—measurement of the intensity of light reflectedfrom a surface where the properties of the surface influences thereflection, e.g. biomolecules which change the refraction index of thesurface.

Polarisation measurement—measurement of the polarisation of polarisedlight, usually as the angle of polarisation, which is depending on thebinding of biomolecules, virus or cells.

Surface plasmon spectroscopy—optical physical measurement techniquewhich utilise the surface plasmon condition of thin metal surfaces,which can be used to measure small changes of refraction index with highsensitivity, e.g. as caused by the presence of biomolecules on thesurface.

Ellipsometry—optical physical measurement technique which can be used tomeasure small changes of refraction index at surfaces with highsensitivity, by measuring changes in elliptisity of polarised light,e.g. as caused by the presence of biomolecules on the surface.

Piezoelectric crystal—crystal which frequency can be influenced bychanges of mass or pressure which can be measured electrically, forexample the change of mass caused by the presence of biomolecule(s),virus or cell(s) bound to the crystal surface.

Electrochemical electrode—measuring device which generates an electricalsignal caused by an electrochemical reaction at the electrode which isrelated to a chemical parameter, e.g. pH, PO₂, pCO₂, the values of whichcan vary because of the presence of analyte(s) in a sample specific fora compound bound to the measuring device.

Thermistor—electrical resistance device which changes resistance withthe temperature; biochemical reactions are characterised by e.g.specific values of heat consumption/formation, which can be registeredvia the thermistor.

A large amount of the carbohydrate sequences present in glycoproteins orin glycolipids, and usually also smaller fragments of these sequences,have shown biospecific binding to proteins, virus or cells.

The present invention describes a biosensor where this specificity isused for determination of such a component in a sample. The invention ischaracterised by that the carbohydrate or a derivative thereof is boundto a surface in the biosensor.

As carbohydrate, one can use fragments (oligosaccharides) of thecarbohydrate sequences found in glycoproteins or in glycolipids and onecan also use smaller fragments of these sequences, i.e. disaccharide,trisaccharide, tetrasaccharide or a pentasaccharide, because this sizeusually is sufficient for the oligosaccharide to bind a protein, virusor a cell in a biospecific manner. A review or such carbohydratesequences can be found in e.g. Chemistry and Physics of Lipids, vol. 42,p. 153-172, 1986, and in Ann. Rev. Biochem., vol. 58, p. 309-350.

The oligosaccharide is usually modified in the reducing end with anaglycon, which is composed of a glycosidically bound organic group whichis suitable for binding to the surface in the biosensor. Examples ofaglycons are OEtSEtCONHNH₂, —OEtSPhNH₂, etc. The binding to the surfacein the biosensor can be done directly or via proteins, e.g. bovine serumalbumine or via a chemical structure which has been adsorbed or whichhas been covalently bound to the surface. Such a chemical structure cancontain reactive organic groups such as carboxyl-, sulfonate, cyanate,epoxy-, aldehyde groups or other groups suitable for chemicalconjugation with for example an amine or thiol group in the aglycon.

More specific examples of analytes which can be analysed with biosensoraccording to the present invention are lectins, antibodies againstcarbohydrates, pathogenic virus or bacteria, such as urinary tractbacteria (e.g. P-fimbriated E. coli) or pathogens of the respiratorytract, and bacteria which cause infections/diarrhea in gastrointestinaltract.

Non-limiting examples of carbohydrate structures of interest and whichcan be used in the form of a carbohydrate derivative in a biosensoraccording to the invention, are monosaccharides, disaccharides,trisaccharides and higher oligosaccharides which show biologicalactivity or which has the ability to specifically bind one or morebiomolecules or a group of biomolecules. Examples of biomolecules areother saccharides, peptides and proteins. Examples of such carbohydratesequences are the blood group determinants (for example A, B, H,Lewis-a, Lewis-b, Lewis-x, Lewis-y), cancer-associated carbohydratesequences, carbohydrate sequences (often di, tri- or tetrasaccharides)which bind to pathogenic bacteria/toxins or virus of for example therespiratory, the gastro-intestinal or the urinary tract, carbohydratesequences which bind to proteins/cells/white blood cells associated withinflammatory reactions (for example selectin-carbohydrate reactions).

These and other carbohydrate structures which can be used in a biosensoraccording to the present invention often contain one or more of thefollowing monosaccharides (or a derivative or an analog of any of these)which are (∝ or β-glycosidically bound: hexosamine, fucose, mannose,glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, xylose,galactose, or another monosaccharide. These components are usuallypresent in for example pyranose or furanose form.

Examples of carbohydrate derivatives are derivatives where thecarbohydrate or a derivative or an analog, are modified in the reducingend with an 0-, N—, C— or S-glycosidically bound aglycon which can be analiphatic or an aromatic compound, an amino acid-, peptide- or proteinmolecule or a derivative thereof. The aglycon part can thus be composedof for example an 0-, N—, C— or S-glycosidically bound aliphatic oraromatic compound which is bound to an amino acid-, peptide- or proteinmolecule or a derivative thereof. Examples of carbohydrate derivativeswhich can be used according to the invention are structures in which oneor more of the hydroxyl groups in the carbohydrate, in addition to orinstead of the hydroxyl group in the reducing end of the carbohydratepart, have been modified with an organic or inorganic group. This can beof interest, for example to increase/modify the biological activity orto facilitate the binding to the biosensor surface according to theinvention.

The aglycon part or another group can be used for adsorption or covalentbinding of the carbohydrate derivative to the surface of the biosensorand can be used in the invention as a spacer molecule between thebiosensor surface and the carbohydrate part to minimise stericalhindrance in the binding of the analyte to the carbohydrate part in thebiosensor according to the invention.

The aliphatic or aromatic compound in the aglycon can for exampleconsist of structures of the type —R—X, where R— consists of an organiccompound, for example an alkyl chain of the type (—CH₂)_(n), in which nis an integer, e.g. in the interval 2 to 8, or is composed of anaromatic group-containing structure, and where —X is for example astructure of the type —S—, amide (—NH—CO— or CO—NH—), amine (—NH—), a—N═N— group or another group suitable for binding to the surface in thebiosensor or to a protein (i.e. in the latter case the carbohydratederivative is a neoglycoprotein). When the carbohydrate derivative is aneoglycoprotein R can be used as a spacer between the protein part andthe carbohydrate part. The spacer often has a functional part (—X—above) which has been used in the binding to the protein.

Suitable spacer and functional group is chosen by the person skilled inthe art and does not limit the scope of the invention.

The carbohydrate derivative can also, according to the invention, becomposed of a natural, in vitro isolated glycoprotein or a recombinantglycoprotein or a glycopeptide. This type of derivative can be adsorbedto the surface in the biosensor, for example a gold- or silica surfaceor another surface which adsorbs proteins, lipids or peptides.

In the case a covalent binding is desired one can, as in the case whenthe carbohydrate derivative is a neoglycoprotein, use for example theprotein part's amino-, carboxyl-, or thiol groups for binding to thesurface in the biosensor. This (as for the synthesis of theneoglycoprotein from carbohydrate spacer and protein) can be done withthe standard techniques which normally are used for modification ofproteins and for immobilisation of proteins to solid supports (see forexample methods mentioned in Methods of Enzymology, volumes 44, 102, and135), and the choice of suitable technology is made by the personskilled in the art in every specific case. Examples of methods arecoupling or activation of carboxyl groups with carbodiimide reagents,N-hydroxysuccinimide reagents, of hydroxyl groups with CNBr, sulphonylchloride (tresyl chloride, tosyl chloride), divinyl sulphone, periodate(gives aldehyde groups), thiol groups are activated with thiol reagentsof the type N-succinionidyl 3-(2-pyridyldithio)propionate, etc.

As examples of surfaces according to the invention may be mentioned:

-   -   Carbohydrate-R—X-Biosensor surface or        Carbohydrate-R—X-Protein-Biosensor surface,    -   where Carbohydrate, R and X have been exemplified above. X and        Protein can be directly adsorbed on the Biosensor surface above,        but between X and Biosensor surface above and between Protein        and Biosensor surface above can also a chemical group be        present, for example a —CO—CH₂CH₂—S-group, i.e. for example:    -   Carbohydrate-R—NH—CO—CH₂—CH₂—S-Biosensor surface or    -   Carbohydrate-R—X-Protein-NH—CO—CH₂—CH₂—S-Biosensor surface.

As protein one can use for example bovine serum albumin, but all for theapplication suitable types of proteins can be used in the carbohydratederivative-based biosensor according to the invention.

The biosensor according to the invention can be designed in a variety ofconfigurations. Examples are:

-   -   a) planar carbohydrate surface which easily can be contacted        with the sample, for example a surface designed as a dipstick,        this surface can be placed in a measuring device for optical        reflectance measurement in air.    -   b) Flow system with flow cell, the surface of which is modified        with carbohydrate and where the signal is transferred with        optical, electrochemical, thermical or gravimetric method and        where the measuring device is placed in, or in close connection        with the cell.    -   c) Cuvettte or other sample cell, which has been connected with        a signal transducer equipped with carbohydrate to which the        sample is added.    -   d) Planar carbohydrate surface which consists of part of the        signal transducer which with ease can be brought into contact        with the sample for a suitable time, whereafter the sample is        removed and the surface of the signal transducer is        characterised with a physical measuring method, for example        electronic measurement, gravimetric measurement or thermal        measurement.

In some situations, e.g. to increase the biosensor signal in themeasurement of low concentrations of cells, it can be advantageous inthe measurement of the analyte with the biosensor to add, after thebinding of the analyte to the carbohydrate surface, micro particlesmodified with carbohydrate specific for the bound cell.

The surface of the biosensor can be, for example a gold surface or amodified gold surface, a plastic surface which has been modified with agold surface, silver surface or another metallic surface, ormodifications thereof with polymers to which chemical coupling ofcarbohydrate can be carried out.

Below are given non-limiting examples of carbohydrate surfaces which canbe used in biosensors according to the invention for binding andanalysis/determination of pathogenic bacteria of the urinary tract.

EXAMPLE

One example was performed as follows: Silica surface coated with a goldlayer was modified with mercaptopropionic acid by dipping the surface ina 5 mM solution of the acid. The carboxyl groups were modified withcarbodiimide (EDC) for 2 hours, whereafter digalactoside with aglycon(Gal ∝ 1-4Galβ-OEtSEtCONHNH₂), was coupled to the EDC-activated surfacefor 12 hours at pH 8.5 and the surface was then rinsed with buffer.

The thus obtained gold surface modified with digalactoside was dippedfor 60 minutes (this time can be varied) in a sample with bacteria ofthe urinary tract (P-fimbriated E. Coli) containing Gal ∝1-4Gal-specific receptor protein, followed by rinsing of the surfacewith distilled water for 2 minutes. Another gold surface modified in thesame way with Gal ∝ 1-4Gal, was dipped in a sample containing anothernon-infectious E. Coli strain which lack the Gal ∝ 1-4Gal-specificreceptor protein. The extent of binding of the different bacteria to thesurfaces was compared with electrom microscopy. The bacteria with theGal 4 1-4Gal-receptor bound to the surface to a ca 10-15 times higherextent than the other bacteria. The binding of P-fimbriated E. Coli to agold surface modified with mercapto-propionic acid alone, was ca 20times lower than to the Gal ∝ 1-4Gal-modified surface.

Alternative non-limiting examples are given below in which aneoglycoprotein was bound covalently or adsorbed diredly on a surfacefor use in biosensor according to the invention.

In procedure B, Gal ∝ 1-4GalβOCH₂CH₂SCH₂CH₂C(O)—NHNH—BSA was coupled tothe same type of EDC-activated gold plate as in the procedure above. TheGalabiose-BSA derivative (0.1 mg/ml) was dissolved in 0.1 M boronate, pH8.5 and EDC-activated plates were immersed in this solution for 1 hour.Subsequently the plates were immersed in a BSA solution (3 mg/ml) inphosphate buffer for 1 minute and rinsed with buffer and distilled waterand stored as above.

In procedure C, Gold plates (not pretreated with mercaptopropionic acid)were immersed in a solution of Gal ∝ 1-4Galβ-BSA (0.1 mg/ml) in 0.1 Msodium phosphate, pH 6.0, for 1 hour and subsequently immersed in theabove BSA solution (3 mg/ml) for 1 minute, rinsed with buffer anddistilled water and stored as above.

These latter biosensor surfaces showed similar characteristics and lowback-ground binding of bacteria as surface in the first example above.

1. An immobilized carbohydrate derivative biosensor, comprising: asurface; and a carbohydrate derivative, bound to the surface, whichspecifically binds to at least one biomolecule in a sample wherein thecarbohydrate derivative comprises a fragment of a carbohydrate sequencefound in a glycoprotein or a glycolipid which fragment is a mono, di,tri, oligo or polysaccharide.
 2. The immobilized carbohydrate derivativebiosensor, according to claim 1, wherein said fragment is modified inthe reducing end with an aglycon which contains at least one protein,aliphatic or aromatic compound.
 3. An immobilized carbohydratederivative biosensor, comprising: a surface; and a carbohydratederivative, bound to the surface by a protein or a chemical structure,which specifically binds to at least one biomolecule in a sample whereinthe carbohydrate is modified in the reducing end with an O—, N—, C— orS-glycosidically bound aglycon.
 4. The immobilized carbohydratederivative biosensor according to claim 3, wherein the glycosidicallybound aglycon comprises a structure corresponding to a formula —R—X,wherein the biosensor corresponds to the formula: carbohydrate-R—Xbiosensor surface, wherein R is aliphatic or aromatic, protein orpolymer, and wherein X is at least one member selected from the groupconsisting of —S—, —NH—CO—, CO—NH, —NH—, and —N═N—.
 5. An immobilizedcarbohydrate derivative biosensor, comprising: a surface; and acarbohydrate derivative, bound to the surface by a member selected fromthe group consisting of carboxyl, sulfonate, cyanate, epoxy andaldehyde, which specifically binds to at least one biomolecule in asample.
 6. The immobilized carbohydrate derivative biosensor accordingto claim 5, wherein the carbohydrate derivative is a derivative in whichthe carbohydrate is modified in the reducing end with an O—, N—, C— orS-glycosidically bound aglycon, comprising a structure corresponding toa formula —R—X, and wherein the biosensor corresponds to the formulacarbohydrate-R—X-protein-NH—CO—CH₂—CH₂—S-biosensor surface, where R isan aliphatic group or an aromatic group.
 7. The immobilized carbohydratederivative biosensor according to claim 5, wherein the protein comprisesbovine serum albumin.
 8. An immobilized carbohydrate derivativebiosensor, comprising: a surface; and a carbohydrate derivative, boundto the surface by a protein or chemical, which specifically binds to atleast one biomolecule in a sample, wherein the surface comprises silicacoated with a gold layer modified with mercaptopropionic acid by dippingthe surface in a 5 mM solution of the acid.
 9. The immobilizedcarbohydrate derivative biosensor according to claim 8, wherein carboxylgroups of said acid are thereafter modified with carbodiimide (EDC),whereafter Galα1-4Galβ-OEtSEtCONHNH₂ is coupled to the surface for 12hours at pH 8.5, and the surface rinsed with a buffer.
 10. A method ofusing the immobilized carbohydrate derivative biosensor according toclaim 9, comprising: dipping the surface in a sample containing bacteriaof the urinary tract having Galα1-4Gal-specific receptor protein;thereafter rinsing the surface with distilled water; and determining theextent of binding of the bacteria to the surface.
 11. The immobilizedcarbohydrate derivative biosensor according to claim 1, wherein thecarbohydrate derivative comprises:Galα1-4GalβOCH₂CH₂SCH₂CH₂C(O)—NHNH—BSA, wherein BSA is bovine serumalbumin.
 12. The immobilized carbohydrate derivative biosensor accordingto claim 1, wherein the carbohydrate derivative comprisesGalα1-4Galβ-BSA, wherein BSA is bovine serum albumin.
 13. Theimmobilized carbohydrate derivative biosensor according to claim 1,wherein the carbohydrate is a member selected from the group consistingof hexosamine, fucose, mannose, glucose, galactose, N-acetylglucosamine, N-acetyl galactosamine and xylose.
 14. The immobilizedcarbohydrate derivative biosensor according to claim 1, wherein theaglycon is bound to an amino acid, peptide or protein.
 15. Theimmobilized carbohydrate derivative biosensor according to claim 1,wherein said carbohydrate derivative is a member selected from the groupconsisting of a natural, in vitro, isolated glycoprotein, a recombinantglycoprotein, a glycopeptide, and neoglycoprotein.
 16. The immobilizedcarbohydrate derivative biosensor according to claim 1, wherein thesurface is a gold, silver, other metallic surface, silica surface, orplastic surface that has been modified with a gold surface, silversurface or other metallic surface or with polymers to which chemicalcoupling of carbohydrate can take place.
 17. The immobilizedcarbohydrate derivative biosensor according to claim 1, wherein thesurface is one which absorbs proteins, lipids or peptides.
 18. Animmobilized carbohydrate fragment biosensor, comprising: a surface; andat least one carbohydrate fragment, bound to the surface, whichspecifically binds to at least one biomolecule in a sample wherein thecarbohydrate derivative comprises a fragment of a carbohydrate sequencefound in a glycoprotein or a glycolipid which is a mono— orpolysaccharide modified in the reducing end with an aglycon whichcontains at least one aliphatic or aromatic compound.
 19. Theimmobilized carbohydrate fragment biosensor according to claim 18,wherein the fragment is a disaccharide, trisaccharide, tetrasaccharideor pentasaccharide.
 20. The immobilized carbohydrate fragment biosensoraccording to claim 18, wherein the aglycon is —OEtSEtCONHNH₂ or—OEtSPhNH₂.
 21. The immobilized carbohydrate fragment biosensoraccording to claim 18, wherein the carbohydrate fragment is bound to thesurface directly, or by a protein or chemical structure which has beenadsorbed or covalently bound to the surface.
 22. The immobilizedcarbohydrate fragment biosensor according to claim 21, wherein thechemical structure is a member selected from the group consisting ofcarboxyl, sulfonate, cyanate, epoxy and aldehyde.
 23. The immobilizedcarbohydrate derivative biosensor according to claim 1, wherein thecarbohydrate derivative is a member selected from the group consistingof monosaccharide, disaccharides, trisaccharides, tetrasaccharides andpentasaccharides.
 24. The immobilized carbohydrate derivative biosensoraccording to claim 1, wherein the carbohydrate derivative has theability to bind to at least one biomolecule.
 25. The immobilizedcarbohydrate derivative biosensor according to claim 24, wherein thebiomolecule is another saccharide, peptide or protein.
 26. Theimmobilized carbohydrate derivative biosensor according to claim 24,wherein the carbohydrate is a member from the blood group determinantsselected from the group consisting of A, B, H, Lewis-a, Lewis-b, Lewis-xand Lewis-y.
 27. The immobilized carbohydrate derivative biosensoraccording to claim 24, wherein the carbohydrate binds to pathogenicbacteria or toxins or virus.
 28. The immobilized carbohydrate derivativebiosensor according to claim 24, wherein the carbohydrate binds toproteins or cells including white blood cells associated withinflammatory reactions.
 29. The immobilized carbohydrate derivativebiosensor according to claim 1, wherein the carbohydrate contains atleast one monosaccharide, derivative or analog thereof which is α orβ-glycosidically bound hexosamine, fucose, mannose, glucose,N-acetyl-glucosamine, N-acetyl-galactosamine, xylose, galactose or othermonosaccharide.
 30. An immobilized carbohydrate derivative biosensor,comprising: a surface; and a carbohydrate derivative, bound to thesurface by a protein or a chemical structure, which specifically bindsto at least one biomolecule in a sample wherein the carbohydrate ismodified in the reducing end with an O—, N—, C— or S-glycosidicallybound aglycon.
 31. The immobilized carbohydrate derivative biosensoraccording to claim 30, wherein the glycosidically bound aglyconcomprises a structure corresponding to a formula —R—X, wherein thebiosensor corresponds to the formula: carbohydrate-R—X biosensorsurface, wherein R is aliphatic or aromatic, a protein or polymer, andwherein X is at least one member selected from the group consisting of—S—, —NH—CO, CO—NH, —NH—, and —N═N—.
 32. The immobilized carbohydratederivative biosensor according to claim 3, wherein the aglycon is anaromatic or aliphatic compound.
 33. The immobilized carbohydratederivative biosensor according to claim 3, wherein the aglycon is anamino acid, peptide or protein, or derivative thereof.
 34. Theimmobilized carbohydrate derivative biosensor according to claim 3,wherein the carbohydrate derivative is a structure in which at least onehydroxyl groups of the carbohydrate in addition to or instead of thehydroxy group in the reducing end of the carbohydrate is furthermodified with an organic or inorganic group.